In urodeles and anurans a major component of retinal regeneration is transdifferentiation of RPE to neural retina. But regeneration is limited in mammals, leaving them susceptible to retinal injury and blinding diseases. Recent studies identified a population of RPE stem cells (RPESC) among cultures of human RPE. These RPESC show unrestricted proliferation and their differentiation potential closely resembled mesenchymal stem cells (MSC), which share a common neuroepithelial origin with RPE. Although RPESC differentiated to express neuronal markers, they failed to induce photoreceptor markers such as RHO. Thus, these RPESC may not represent an intermediate in transdifferentiation of mammalian RPE into photoreceptors. We examined cultures of adult mouse RPE for cells with the differentiation capacity of RPESC, but we failed to identify such cells. However, we found that cells with properties of RPESC can be efficiently and stably induced from RPE (iRPESC) through a hypoxia-dependent pathway, similar to that described for maintenance and induction of MSC. Hypoxia causes RPE damage and is linked to neovascularization and AMD. Key to this iRPESC reprogramming pathway is superinduction of hypoxia inducible factor 1a (Hif1a) to a threshold sufficient to bind and activate the Oct4 stem cell gene promoter. Oct4 in turn induces Dnmt1 which silences cell cycle blocking cyclin dependent kinase inhibitors leading to unrestricted proliferation. These iRPESC are resistant to hypoxia, and importantly, as opposed to human RPESC, they differentiate into Rho+ cells-indeed this differentiation to Rho+ cells is more efficient than seen with embryonic stem cells or induced pluripotent stem cells. Furthermore, iRPESC do not undergo the typical epithelial-mesenchymal transition (EMT) seen when RPE are placed in culture, providing the potential for retaining an RPE phenotype as the cells are expanded. Because blinding diseases such as AMD are highlighted by loss of both functional RPE and photoreceptors, the ability of the iRPESC to undergo photoreceptor differentiation and to resist EMT-initiated loss of phenotype suggest a unique therapeutic potential for the cells. During the two year period of this R21 proposal, we aim to investigate the iRPESC reprogramming pathway on a molecular level. The purpose of these studies is to provide a foundation for experiments designed to optimize photoreceptor differentiation from iRPESC and to maintain a function RPE phenotype as iRPESC are expanded, so in the future we can begin testing the effectiveness of the differentiated iRPESC in transplantation experiments. A second point of this molecular analysis is to identify pathway markers that can ultimately be used for detection of iRPESC in vivo, and to understand factors that might be used in the future to stimulate iRPESC generation from RPE in situ.